Anticonvulsants suppress c-Fos protein expression in spinal cord neurons following noxious thermal stimulation

EXPERIMENTALNEUROLOGY

132.271-278

(1995)

Anticonvulsants Suppress c-Fos Protein Expression in Spinal Cord Neurons Following Noxious Thermal Stimulation THOIVM R. TULLE,*

JOSE M. CiwrRo-Loms,t

JAN SCHADRACK,*

GERARD EVAN,$

AND WALTER

ZIEGLG~NSBERGER*

*Mo.x-Planck-Institute of Psychiatry, Clinical Institute, Clinical Neuropharmacology, Munich, Germany; tlnstitute of Histology and Embryology, Faculty of Medicine of Oporto, Porto, Portugal; and *Imperial Cancer Research Fund Laboratories, St. Bartholomew Hospital, London, England

The expression of the nuclear immediate-early geneencoded protein c-Fos in spinal cord dorsal horn neurons of the rat following noxious thermal stimulation was compared in carbamasepine-, valproate- and phenytoine-treated animals. Single intraperitoneal injection of carbamasepine (50 mg/kg), valproate (300 mg/ kg) or intravenous injection of phenytoine (20 mg/kg) before noxious stimulation reduced the number of c-Fos immunoreactive neurons to 63-60% of control levels in superficial laminae and to 30-60% in deep laminae of the dorsal horn. Pretreatment with carbamasepine or valproate for 4 or 8 days combined with an injection immediately before noxious stimulation further significantly decreased the number of c-Fos neurons in the deep dorsal horn only in animals treated with valproate. The observation that activity-dependent gene expression in the spinal cord is effectively modulated by anticonvulsants discloses a novel therapeutic potential of these compounds. Presumably via an acute suppression of high-frequency repetitive firing and/or altered synaptic transmission of intraspinal or descending neurotransmitter systems these drugs gain access to neuroplastic mechanisms which might be relevant for the restoration of physiological levels of neuronal excitability in the central nervous system. 0 IS95 Academic

F’ress, Inc.

INTRODUCTION

The induction of target genes by transcription factors that are encoded by immediate-early genes (IEGs) (20, 56) can be triggered by synaptic excitation. Such shortterm alterations of excitability are reflected in rapid changes in neuronal .discharge activity and most commonly result from transmembrane ion fluxes. The proto. ncogene c-fos, which is a member of one family of these IEGs is rapidly and transiently induced in neurons of the spinal cord of the rat following noxious stimuli applied to superficial and deep tissue (1, 30, 34, 50, 74, 81). It has been suggested that IEG expression is a prerequisite for the ability of neurons to convert shortterm synaptic stimulation into long-term adaptive regu271

lations of target-genes and, in the spinal cord, may thus induce functional and structural changes that participate in the development or maintenance of chronic pain (13, 17,20,26,56,60). The multiple neurotransmitter and neuromodulator systems which participate in the integration of somatosensory afferents in the dorsal horn of the spinal cord make this structure a promising target for selective pharmacotherapy. Excitatory and inhibitory amino acid transmitters, monoamines and a plethora of neuropeptides have been demonstrated in primary afferents, intrinsic spinal neurons, and descending pathways (for review 8, 84, 85). Opioid agonists, NMDA-antagonists, or a-adrenergic receptor antagonists reduce c-Fos expression in the spinal cord in response to different noxious somatic or visceral stimuli (2, 9, 27, 29, 36, 64, 73, 75, 76). The anticonvulsants carbamazepine, valproate and phenytoine (72) are also therapeutically beneficial in certain forms of chronic pain, such as trigeminal neuralgia or phantom limb pain (41, 57). These pain states readily respond to carbamazepine or phenytoine treatment (22, 23, 57, 66, 82). Anticonvulsants completely abolish the expression of c-Fos in cortex, hippocampus and limbic structures following drug-induced seizures (16,531. In the present study we compared the effects of different anticonvulsants on the expression of c-Fos protein in dorsal horn neurons following noxious stimulation. Since the pharmacological profile of anticonvulsants is supposed to involve rather immediate effects as well as long-term effects, such as enzymatic modulation of the GARA system (37,471, carbamaxepine andvalproate were investigated following single and repetitive application. MATERIAIS

AND METHODS

Experiments were performed with 33 male Animals. Sprague-Dawley rats weighing 230-280 g. Five days before the experiment or pretreatment the animals were single housed with food and water ud Zibitum. Experimental and pharmacological treatment. Animals were anesthetized by spontaneously breathing a 0014-4899/95 $9.00 Copyright 6 1995 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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mixture of 1.5 ~01% halothane and 70% ambient sir/30% oxygen. For noxious stimulation the left hind paw was immersed into 52°C hot water (10 times for 20 s with 90-s intervals). Drug-pretreated animals W = 3/4 each experimental group) received carbamazepine (Tegretal, 50 mg/kg ip) or valproate (Ergenyl, 300 mg/kg ip) (i) 40 min before noxious stimulation (NS), (ii) 150 min plus 40 min before NS, (iii) for 4 days once a day and 40 min before NS, or (iv) for 8 days once a day and 40 min before NS. The dosage of carbamazepine and valproate applied in the present study were adopted from studies showing changes of GABA concentrations in the brain following repetitive intraperitoneal injection for 7 days (see 31). The application regime is depicted in Fig. 1. Phenytoine (20 mg/kg) was injected intravenously (external jugular vein) 10 min before noxious stimulation in another group of animals. In control animals (N = 9) saline was injected under the same conditions at the corresponding time points. After termination of the stimulation the anesthesia was changed to 0.8 ~01% halothane until the animal was sacrificed 2 h later and perfused with sodium phosphate-buffered saline, followed by 4% paraformaldehyde in phosphate buffer. Blood samples were taken ‘shortly before perfusion to measure the plasma concentration of carbamazepine and valproate. ~mmunoqtochemistry. The spinal cord lumbar enlargements were postfixed for 12 h and washed for 48 h in phosphate buffer with 30% sucrose. For each animal, cryostat coronal serial sections (25 km) were obtained from spinal cord lumbar segments LZ to Si, and every 6fth section was incubated free-floating with a polyclonal antibody against the c-Fos nuclear protein. The antibody was raised in rabbits immunized with a syn.

1 I I I I I , I -0

-1

-6

-5

-4

-3

-1

-1 b

FIG. i&&ions thermal

1. Mode (ip) for stimulation;

I

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-lm

-40 0

I 140 laimlks

and frequency’ of carbamazepine and valproate the different drug-treatment groups. NS, noxious Perfusion, intracardial perfusion.

thetic peptide containing sequences common to the N-terminal region of all known c-Fos and v-Fos proteins (for details see Ref. 34). Sections were preincubated in normal goat serum (2% in phosphate-buffered saline and 0.2% Triton X-100) for 2 h followed by the primary antisera, diluted 1:3000, for 12-18 h at 20°C. Immunoreactivity was visualized with an avidin-biotin-HRP method (Vectastuin, Vector Laboratories, USA). Sections were developed in 0.02% diaminobenzidine (DAB) with 0.02% hydrogen peroxide. Prior incubation of antiserum with 10 pg of native peptide completely abolished all immunoreactivities. Because of the possibility of cross-reactivity, the observed immunoreactivity should be regarded as “peptide-like immunoreactivity.” Control animals were always processed simultaneously with drug-treated animals to ensure the quality of the immunostaining technique and to serve as same day control animals for calculation of relative measurements. Tissue secQuantification and statistical analysis. tions from lumbar segments Lz-Si were examined with bright-field microscopy to identify immunoreactive neurons. Regardless of staining intensity, ranging from lightly brown to dark brown/black, neurons were termed “IEG-positive” if the nuclei showed the characteristic staining of oxidized DAB and a clear distinction from the background in low and high magnifications (4x, 10x, and 20 x 1. For all animals the highest number of IEGpositive neurons was found in the lumbar segments Lp5. The segmental level was verified with dark-field illumination (52). For each animal the 5 most stained sections from approximately 30 sections from the Lp5 region were quantified for the number of stained nuclei in the superficial dorsal horn (sDH; laminae I-11,111) and the deep dorsal horn (dDH; laminae IV-V-LX). Counterstaining with cresyl-violet and viewing against a standard plan with the boundaries of the spinal laminae (63) was used to assign the immunostained neurons to Rexed’s laminae. For statistical analysis the average number of IEGpositive neurons over the five most stained sections was calculated. Counts are expressed as the average number of Fos-positive neurons per section (n/s) or as percentage change of total number of neurons per section + SEM, relative to the averaged number of neurons of control animals (i.e., animals stimulated with noxious heat without pharmacological treatment) processed in the same series. For testing the effects of the frequency of drug application and region (sDH, dDH) on Fospositive neurons a two-way analysis of variance (ANOVA) was performed for carbamazepine- and for valproatetreatment. The differences between the various drugtreatment groups and the control group were tested about significance for both anatomical regions with the Duncan post hoc test in a followed one-way ANOVA. As level of significance OL= 0.05 was accepted. This level has been adjusted (Bonferro,ni procedure) when and

IMMEDIATE-EARLY

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EXPRESSION

where post hoc tests had to be carried out. Data concerning the animals treated with phenytoine are presented with descriptive statistics. During the experimental procedures and counting-phase, the investigators were blind to the treatment of the animals. RESULTS

Anatomical

Pattern of c-Fos Expression

In unstimulated rats that were not exposed to the noxious stimulus only a few scattered neurons immunoreactive for c-Fos could be detected in some sections of the sDH. Counterstaining revealed that the immunoreactive material was located in the nucleus of the neurons. Two hours following noxious heat stimulation numerous neurons with nuclei expressing c-Fos protein were found distributed mainly in the medial half of the ipsilateral lamina I-II extending from lumbar segment Lz-Si with a maximum around LkS. Stained neurons occurred also in deeper laminae (III-VI and X) along their mediolateral extension, although in smaller numbers (Fig. 2A). The distribution of c-Fos expressing neurons in the spinal cord dorsal horn overlapped with the known central terminations of primary afferent fibers from the hind-limb (71). The average number of stained neurons in the most stained slices was approximately 80 (n/s) in superficial laminae and about 30 (n/s) in deeper laminae (Fig. 2A). There was little interindividual variability both in the number and the pattern of distribution of neurons. Only occasionally, a few scattered c-Fos positive neurons were observed in the contralateral half of the spinal cord. Pharmacological

Modulation

of c-Fos Expression

Carbamazepine, valproate, or phenytoine, as well as intravenous cannulation of the jugular vein, did not influence the basal c-Fos expression in unstimulated control animals (not shown). Following single injection of carbamazepine 40 min before noxious stimulation the number of c-Fos positive neurons was reduced to 78 * 1% in sDH and to 28 2 6% in dDH (Figs. 2B and 31 as compared to noxious heat-stimulated control animals. The combined application 150 and 40 min before NS reduced the amount of c-Fos positive neurons to 75 * 7% in sDH and 32 * 5% in dDH. Repetitive injection of carbamazepine for 4 or 8 days and an application 40 min before NS suppressed the number of c-Fos positive neurons to 71 + 4% in sDH and 31 -t 10% in dDH and to

FIG. 2. Photomicrographs of the ipsilateral dorsal horn of c-Fosimmunostained lumbar spinal cord slices following noxious thermal stimulation of one hind foot (A). (B) Pretreatment with carbamazepine (50 mg/kg ip). (C) Pretreatment of the animal for 8 days with valproate (300 mg/kg ip) in addition to an injection 40 min before noxious stimulation. Bar in (0, 100 grn.

IN THE

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65 f 6% in sDH and 17 + 4% in dDH, respectively (Fig. 3). For valproate the corresponding values showed a reduction of oFos positive neurons to 62 + 5% in sDH and 60 f 15% in dDH after a single injection 40 min before NS (Fig. 4). Application of the anticonvulsant 150 and 40 min before noxious stimulation produced a suppression of c-Fos positive neurons to 76 + 4% of control in sDH and 41 2 5% in dDH. Pretreatment of the animals with daily injections of valproate in addition to an injection immediately before the noxious stimulation reduced the number of c-F06 positive neurons to 73 f 1% in sDH and 49 + 9% in dDH (4 days pretreatment) and to 53 + 2% in sDH and 22 + 3% in dDH (8 days pretreatment) (Figs. 2C and 4). For both, carbamazepineand valproate-treatment two-way analysis of variance showed a significant main effect of drug-treatment and a significant main effect of region, as well as a significant drug-treatment n region interaction, indicating that the effect of pharmacological treatment varied between the sDH and dDH (Table 11. The one-way analysis of variance revealed a significant effect of carbsmazepine-as well as of valproate-treatment both in sDH and in dDH (Table 1). In all cases the number of c-F06 positive neurons in the drug-treatment groups was significantly less compared to control groups (post hoc Duncan tests, P < 0.05). For valproate, the level of c-Fos suppression increased in the dDH with the frequency of application, showing a significant difference between 8 days of treatment compared to a single application (post hoc Duncan test, P < 0.05) (Fig. 4). For carbamasepine, however, the mean values of relative changes for Fos-positive neurons in sDH and dDH showed no differences in the effectiveness of c-F06 . suppression following repetitive application. Single intravenous application of phenytoine suppressed c-F06 expression in sDH to 76 + 4% and 58 k 3% in dDH. In addition to the overall reduction of the number of c-F06 % of con1rol

”

I

conool (saline)

4oO’

-150’ -40’

-4 days -40’

-8 “2

FIG. 3. Expression of c-F06 immunoreactivity in spinal cord neurons of laminae I-II (open bars) and laminae III-VI,X (hatched bars) following noxious stimulation and pretreatment with carbamaaepine (50 mg/kg ip) for different ‘durations. Values given represent group specific mean and SEM of c-F08 positive neurons relative to their corresponding saline control values (100%).

ET AL. % of conrrol

I

EOlltrOl

.^.

-4u.

(saline)

-4 days -40’

-8 days 40’

FIG. 4. Expression of c-F08 immunoreactivity in spinal cord neurons of laminae I-II (open bars) and laminae III-VI,X (hatched bars) following noxious stimulation and pretreatment with valproate (300 mglkg ip) for different durations. Values given represent group specific averages and SEM of c-Fos positive neurons relative to their corresponding saline control levels (100%).

positive neurons, neurons in animals treated with the anticonvulsants occasionally showed a lighter nuclear immunostaining than controls (not shown). No attempt has been made to quantify this lighter staining with relative optical density measurements. Calculations of the carbamaxepine and valproate plasma levels revealed 12 k 5.6 kg/ml for carbamazepine and 267 + 64.40 pg/ml for valproate. The plasma levels attained for these two compounds are above those necessary for antiepileptic treatment in humans. No significant differences between plasma levels following short-term or long-term application of the drugs were detected. DISCUSSION

The present study is in accord with previous findings that noxious thermal stimulation induces c-Fos expression in spinal cord dorsal horn neurons (30,34, 79,811. The acute application of anticonvulsants effectively suppresses c-F06 expression and, moreover, pretreatment with valproate over a longer period of time further enhances this suppressive effect in deep dorsal horn neurons. The expression of c-fos in the mammalian nervous system is closely linked to the intracellular calcium (Ca’+J concentration (55, 56). Ca2+-influx via voltagedependent calcium channels, Ca2+-channel activating drugs (54) or activation of excitatory amino acid (EAA) receptors (42) may serve as the intracellular signal coupling synaptic activation with gene expression. Ionotropic glutamate receptor subtypes activate c-fis transcription probably by distinct Ca2+ requiring intracellular signaling pathways (4,431. In the spinal cord the increase of Ca2+i in dorsal horn neurons is assumed to result from the release of EAAs, such as L-glutamate, and various peptide neurotransmit-

IMMEDIATE-EARLY

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IN THE

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TABLE 1

Results (F-Values and Corresponding Significances) for Two- and One-Way Analysis of Variance (ANOVA) Performed for the Number of c-Fos Positive Neurons Factors

ANOVA Two-way

One-way

ANOVA

ANOVA

Note. The considered and anatomical region

Carbamazepine

Drug-treatment Region Interaction Drug-treatment Drug treatment

(region (region

= sDH) = dDH)

F(4,36) F(1.36) F(4,36) F(4,18) F(4,181

= = = = =

32.484; sig of F < 0.00001 48.413; sigofF < 0.00001 6.205; sig of F < 0.00001 6.8634; sigofF < 0.00001 27.4032; sig of F < 0.00001

factors were drug-treatment (carbamazepine 50 mg/kg in the spinal cord of the rat (superficial vs deep laminae).

ters, such as substance P (SP) from primary afferent fibers following peripheral noxious stimulation (18). Since the amount of c-Fos protein produced by dorsal horn neurons is proportional to the degree of transsynaptic activation (10, 34, 79) the suppressive effect of carbamazepine, valproate, and phenytoine on c-Fos expression is likely to emerge from the depression of neuronal discharge activity and the concomittant decrease in Ca2+i. The physiological mechanisms by which anticonvulsants can achieve this effect include (i) suppression of high-frequency repetitive firing by acting on the conductance of various ion channels (5, 21, 25, 77), (ii) postsynaptic potentiation of GABA responses or a decrease of excitatory synaptic transmission and regulation of Ca2+ dependent neurotransmitter release (14, 15), and (iii) an augmented GABAergic inhibition by activation of blockage of enzymes involved in GABAmetabolism (35,37,45,47,69). Phenytoine and carbamazepine reduce polysynaptic reflexes and post-tetanic potentiation of monosynaptic reflexes in the spinal cord (19,39) and increase the latency of synaptic responses in single neurons of the trigeminal nucleus in uiuo (23). The analgesic effects of anticonvulsant drugs in the treatment of head and face pain frequently occur only after treatment for several days or weeks (22, 72). In spinal cord neurons in vitro, phenytoine, carbamazepine and valproate, at concentrations equivalent to therapeutic plasma levels, produce a voltage-dependent limitation of sustained repetitive firing without changing resting membrane properties and, at least in the majority of neurons, the postsynaptic responses to iontophoretically applied GABA and glutamate (46, 48, 49). N-methyl-n-aspartate (NMDA)-activated currents are dose-dependently blocked by carbamazepine, but not by phenytoine (40). There is increasing evidence that the . NMDA subtype of the glutamate ionotropic receptor is involved in polysynaptic nociceptive transmission in the spinal cord (28) and that the modulatory effect of NMDA-antagonists on c-Fos expression depends on the quality of the peripheral noxious stimulus (9, 38, 73, 81). As c-Fos expression by noxious thermal stimulation (73, 81) is not affected by NMDA-antagonists, in the

Valproate

ip or valproate

300 mg/kg

F(4,36) F(1,36) F(4,36) F(4,18) F(4.18)

= = = = =

ip at different

30.342; sigof F < 0.00001 17.004; sigofF < 0.00001 3.362; sig of F < 0.00001 14.7733; sig of F < 0.00001 17.7133; sig of F < 0.00001 frequencies

of injections)

present study, the suppressive effects of carbamazepine are probably not exerted via NMDA-receptor modulation (83). Beside effects on central neuronal excitability, in mammalian myelinated nerve fibers, carbamazepine and phenytoine reduce the conduction velocity and the amplitude of the compound action potential (32,39) and block potential- and frequency-dependent Na+ currents (70). Thus, a diminution of primary afferent, transsynaptic activation of dorsal horn neurons following peripheral noxious stimulation may contribute to the suppression of c-Fos expression. This “membrane-stabilization” of peripheral nerves and depression of impulse propagation may contribute to the therapeutic effects of anticonv&ants on neuralgic pain (57). The long-term treatment with anticonvulsants affects the metabolism of several neurotransmitter systems, including that of GABA (35). While phenytoine has no effects on the level of GABA in the brain (62), valproate increases GABA content in synaptic terminals (44) and various brain structures (31) by activation of the GABA synthesizing enzyme glutamate decarboxylase (GAD) (44,58). Carbamazepine changes brain GABA concentration (7, 31) probably by inhibition of the GABA degrading enzyme succinic semialdehyde dehydrogenase (67). Segmental and supraspinal GABA systems are both implicated in central pain modulation (68). High levels of GABA and its synthesizing enzyme GAD as well as immunoreactivity for anti-GAD and anti-GABA are found in the superficial dorsal horn of normal rats (3, 33, 51). Enhanced concentrations of GAD mBNA and GABA are found in the spinal cord following inflammation ( 11,12). The involvement of GABA-mediated mechanisms in spinal antinociception (68, 80) is further evidenced by the modulation of c-Fos expression in the spinal cord following noxious inflammation by the prototypic GABAs agonist baclofen (6; T. R. T6lle et al., unpublished observations). It awaits further investigations whether the more effective suppression of c-Fos expression following long-term application of valproate results from an-valproate mediated+nbanced GABAergic inhibition of spinal cord neurons by de nouo

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GABA synthesis, reduced degradation (67), or a changed interaction between carbamasepine and GABA receptor complexes (61). The present data suggest that anticonvulsants are able to prevent long-term changes induced by activitydependent gene modulation not only in the hippocampus during seizures (24,59,78). The maintained suppression of discharge activity in neurons involved in pain signaling by anticonvulsants could help to restore physiological discharge activity in these neurons by extinction of neuroplastic adaptive mechanisms and help to alleviate pain and probably also spasticity.

ET

15.

16. 17. 18.

REFERENCES

1. ABBADIE, C., AND J. M. BE.SSON.1992. c-Fos expression in rat lumbar spinal cord during the development of adjuvant-induced arthritis. Neurvscience 48: 985-993. 2. A~BADIE, C., AND J. M. BESSON. 1993. Effects of morphine and naloxone on basal and evoked Fos-Iike immunoreactivity in lumbar spinal cord neurons of arthritc rata. Pain 52: 29-39. of 3. ALBERS, R. W., AND R. 0. BRADY. 1959. The distribution glutamic decarboxyiase in the nervous system of the rhesus monkey. J. Bial. C&m. 234: 926-928. 4. BADING, H., D. D. GINTY, ANDM. E. GREENBERG.1993. Regulation of gene expression in hippocampal neurons by distinct calcium signahngpathways. Science 266r 181-186. 5. BAKER, P. F., A. C. HODGKIN, ANII E. F. RIDCWAY. 1971. Depolarization and calcium entry in squid giant axons. J. Physiol. (Landon) 218: 709-755. 6. BASBAUM, A I., R. PRESLEY, D. MENETREY, S.-I. CHI, AND J. D. LEVINE. 1989. Somatic, at-tic&r and visceral noxious-stimulus evoked expression of c-Fos in the spinal cord of the rat: differential patterns of activity and modulation by analgesic agents. Pages 365-381 in F. Cervero, G. J. Bennett, and P. M. Headley, Eds., Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Vol. 176. Plenum Press, New York. AND G. ROMAN. 1984. 7. BATTISTINI, L. M., M. VAROTTO,G. BE-E, Effects of some anticonvuisant drugs on brain GABA level and GAD and GABA-T activities. Neurochem. Res. 9: 225-231. 8. @SSON, J. M., AND A CHAOUCH. 1987. Peripheral and spinal mechanisms of nociception. Physiol Rev. 67: 67-154. 9. BIRDER, L. A., AND W. C. DE GROAT. 1992. The effect of glutamate antagonists on c-F06 expression induced in spinal neurons by irritation of the lower urinary tract. Brain Res. 580: 1 X-120. 10. BULLITT, E., C. L. LEE, A. R. LIGHT, AND H. WILLCCCKSON. 1992. The effect of stimulus duration on noxious-stimulus induced c-Fos expression in the rodent spinal cord. Brain Res. 580: 172-179. 11. CASTRO-LOPES,J. M., I. TAVARES,T. R. T&E, A. COITO, AND A. Comrm4.1992. Increase in GABAergic cells and GABA levels in the spinal cord in uniiateral inilammation of the hindiimb in the rat. Eur. J. Neurvsci. 4: 296-301. 12. CASTRO-LOPES,J. M., T. R. TULLE, B. PAN, AND W. ZIEGLC~~SBERGER.1994. Expression of GAD mRNA in spinal cord neurons of normal and monoarthritic rata. Mol. Brain. Res. 26: 169-176. 13. CODE-, T. J., J. KATZ, A. L. VACCARINO,ANDR. MEL&+&K. 1993. Contribution of central neuroplasticity to pathological pain: review of chnicai and experimental evidence. Pain 52: 259-285. 14. DELORENZO,R. J. 1980. Antiepileptic drugs. Phenytoin: caiciumand cahnoduhn-dependent pmtein phosphoryiation and neurotransmitter release. Pages 399-414 in G. H. GIaser, J. K. Penry,

AL.

and D. M. Woodbury, Eds., Antiepileptic Drugs: Mechanism of Action Raven Press, New York. DELORENZO, R. J. 1986. A molecular approach to the calcium signal in brain: relationship to synaptic modulation and seizure discharge. Pages 435-464 in A. V. Delgado-Escueta, A. A. Ward, Jr., D. M. Woodbury, and R. J. Porter, Eds., Advances in Neurology, Vol. 44. Raven Press, New York. DRAGUNOW,M., AND H. A. ROBERTSON. 1988. Localization and induction of c-Fos protein-like immunoreactive material in the nuclei of adult mammalian neurons. Brain Res. 440: 252-260. DUBNER, R., AND M. A. RUDA. 1992. Activity-dependent neuronai plasticity following tissue injury and inflammation. Trends Neurasci. 15: 96-103. DUGCAN,A. W., I. A. HENDRY,C. R. MORTON,W. D. HUTCHINSON, AND Z. Q. ZHAO. 1988. Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat. Brain Res. 451: 261-273.

19.

20. 21.

22. 23.

ESPLIN, D. W. 1957. Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. J. Pharmacol. Exp. Ther. 120: 301-323. EVAN, G. 1991. Regulation of gene expression. Br. Med. Bulletin 47: 116-135. FERRENDELLI,J. A., AND S. DANIELS-MCQUEEN. 1982. Comparative actions of phenytoin and other anticonvulsant drugs on potassium- and veratridine-stimulated calcium uptake in synaptosomes. J. Pharmaeol. Exp. Ther. 220: 29-34. FROMM, G. H. 1990. Clinical pharmacology of drugs used to treat head and face pain. Neural. Clin. 8: 143-151. FROMM, G. H., ANDJ. M. KILL~~W. 1967. Effect of some anticonvulsant drugs on the spinal trigeminai nucleus. Neurology 17: 275-280.

24:

25.

26.

27.

28.

29.

GALL, C., J. LAUTERBORN, M. BUNDMAN, K. MURRAY, AND P. ISACKSON. 1991. Seizures and the regulation of neurothrophic factor and neuropeptide gene expression in brain. Pages 225-245 in V. E. Anderson, W. A. Hauser, I. E. Leppik, J. L. Noebels, and S. S. Rich, Eds., Genetic Stmtegies in Epilepsy Research (Epilepsy Res. Suppl. 4) Elsevier, Amsterdam. GASSER, T., M. REDDINGTON,AND P. SCHUBERT. 1988. Effect of csrbamasepine on stimulus-evoked Ca2+ fluxes in rat hippocampai slices and its interaction with Al-adenosine receptors. Neurosci. Lett. 91: 189-193. GOELET, P., V. F. CASTELLUCCI,S. SCHACHER,AND E. R. KANDEL. 1986. The long and the short of long-term memory-a molecular framework. Nature 322: 419-422. GOGAS, K. R., R. W. PRESLEY, J. D. LEVINE, AND A. I. BASBAUM. 1991. The antinociceptive action of supraspinai opioids results from an increase in descending inhibitory control: Correlation of nociceptive behavior and c-Fos expression. Neuroscience 42: 617-628. HEADLEY, P. M., AND S. GRILLNER. 1990. Excitatory amino acids and synaptic transmission: the evidence for a physiological function. TrendsPharmacol. Sci. 11: 205-211. HAMMOND, D. L., R. W. PRESLEY,K. R. GOGAS,ANDA. I. BASBAUM. 1992. Morphine or U-50,488 suppresses Fos protein-like immunoreactivity in the spinal cord and nucleus tractus solitarii evoked by a noxious visceral stimulus in the rat. J. Comp. Neurol. 315:

30.

244-253.

HERDEGEN,T., T. R. TULLE, R. BRAVO, W. ZIEGLG~~NSBERGER, AND M. ZIMMERMANN. 1991. Sequential expression of Jun B, Jun D and Fos B proteins in rat spinal neurons: cascade of transcrip tionai operations during nociception. Neurosci. Wt. 129: 221224.

31.

HIGUCHI, T., 0. YAMAZAKI, A. TAKAZAWA,N. KATO, N. WATANABE, Y. MINATOGAWA,J. YAMAZAKI, H. OHSIUMA, S. NAGAKI, Y. IGARASHI,AND T. NOCUCHI. 1986. Effects of carbamaxepine and vaiproic

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32. 33.

34. 35.

36. 37.

38.

39. 40. 41.

42.

43. 44. 45.

46. . 47.

48.

GENE EXPRESSION

acid on brain immunoreactive somatostatin and gamma-aminobutyric acid in amygdaloid-kindled rats. Eur. J. Pharm~col. 125: 169-175. 49. HONDA, H., AND M. B. ALLEN. 1973. The effect of an iminostilbene derivative (G 32883) on peripheral nerves. J. Med. Assoc. Ga. 62: 38-42. HUNT, S. P., J. S. KELLY, P. C. EMSON,J. R. KIMMEL, R. J. MILLER, ANDJ.-Y. WV. 1981. An immunohistochemical study of neuronal 50. populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn. Neuroscience 6: 1883-1898. HUNT, S. P., A. PINI, AND G. EVAN. 1987. Induction of c-Fos-like proteins in spinal cord neurons following sensory stimulation. 51. Nature 328: 632-634. JONES, G. L., AND D. M. WOODBURY.1985. Biochemistry. Pages 245-263 in H.-H. Frey, and D. Janz, Eds., Handbook of Experi52. mental Pharmacology. Antiepileptic Drugs, Vol. 74. Springer, Berlin/Heidelberg/New York/Tokyo. JONES, S. L. 1992. Noradrenergic modulation of noxious heat53. evoked Fos-like immunoreactivity in the dorsal horn of the rat spinal cord. J. Comp. Neurol. 325: 435-445. JURNA, I. 1985. Electrophysiological effects of antiepileptic drugs. 54. Pages 611-658 in H.-H. Frey, and D. Janz, Eds., Handbook of Experimental Pharmacology. Antiepileptic Drugs, Vol. 74. 55. Springer, Berlin/Heidelberg/New York/Tokyo. KEHL, L. J., K. R. GOGAS, L. LICHTBLAU, C. H. POLLOCK, M. MAYES, A. I. BASBAUM, AND G. L. WILCOX. 1991. The NMDA 56. antagonist MK-801 reduces noxious stimulus-evoked Fos expression in the spinal cord dorsal horn. Pages 307-311 in M. R. Bond, J. E. Charlton, and C. J. Woolf, Eds., Proceedings of the VZth 57. Congress of Pain. Elsevier, Amsterdam. KRUPP, P. 1969. The effect of TegretahRl on some elementary neuronal mechanisms. Headache 9: 42-46. 58. hhfPE, H., AND H. BIGALKE. 1990. Csrbamszepine blocks NMDAactivated currents in cultured spinal cord neurons. NeuroReport 1:26-28. LENZ, F. A., H. C. KWAN, J. 0. DOSTROVSKY,AND R. R. TASKER. 59. 1989. Characteristics of the bursting pattern of action potentials that occur in the thalamus of patients with central pain. Brain Res. 496: 357-360. 60. LEREA, L. S., L. S. BUTLER, AND J. 0. MCNAMARA. 1992. NMDA and non-NMDA receptor-mediated increase of c-fos mRNA in dentate gyms neurons involves calcium influx via different routes. J. Neurosci. 12: 2973-2981. 61. LERJZA,L. S., AND J. 0. MCNAMARA. 1993. Ionotropic glutamate receptor subtypes activate c-fos transcription by distinct calciumrequiring intracellular signaling pathways. Neuron 10: 31-41. 62. LGSCHER,W. 1981. Valproate induced changes in GABA metabolism at the subcellular level. Biochem. Pharmacol. SO: 136P 1366. L&CHER, W. 1985. Valproic acid. Pages 508-536 in H.-H. Frey, and D. Janz, Eds., Handbook of Experimental Pharmacology. 63. Antepileptic Drugs, Vol. 74. Springer, Berlin/Heidelberg/New York/Tokyo. 64. MACDONALD, R. L., AND G. K. BERGEY. 1979. Valproic acid augments GABA-mediated postsynaptic inhibition in cultured mammalian neurons. Bruin Res. 170: 558-562. MACDONALD, R. L., AND M. J. MCLEAN. 1986. Anticonvulsant 65. drugs: mechanism of action. Pages 713-736 in A. V. DelgadoEscueta, A. A. Ward, and D. M. Woodbury Wheal, Eds., Basic Mechanisms of the Epilepsies: Molecular and Cellular Approaches; Adv. Neurol, Vol. 44. Raven Press, New York. 66. MCLEAN, M. J., AND R. L. MACDONALD.1986a. Sodium valproate, but not ethosuximide, produces use- and voltage-dependent limitation of high frequency repetitive firing of action potentials

IN THE SPINAL

CORD

277

of mouse central in cell culture. J. Pharmacol. Exp. Ther. 297: 1001-1011. MCLEAN, M. J., AND R. L. MACDONALD. 1986b. Carbamazepine and 10,l I-epoxycarbamasepine produce use- and voltage-dependent limitation of rapidly firing action potentials of mouse central neurons in cell culture. J. Pharmacol. Exp. Ther. 238: 727-738. MENETREY,D., A. GANNON,J. D. LEVINE, ANDA. I. BASBAUM. 1989. The expression of c-Fos protein in presumed nociceptive intemeurons and projection neurons of the rat spinal cord: Anatomical mapping of the central effect of noxious somatic, articular and visceral stimulation. J. Camp. Neurol. 285: 177-195. MNATA, Y., ANDM. OTSUKA. 1975. Quantitative hi&chemistry of gamma-aminobutyric acid in cat spinal cord with special reference to presynaptic inhibition. J. New-o&em. 25: 239-244. MOLANDER, C., Q. Xv, AND G. GRANT. 1984. The cytoarchitechtonic organization of the spinal cord in the rat. I. The lower thoracic and lumbosacral cord. J. Comp. Neurol. 230: 133-141. MORGAN, J. I., D. R. COHEN, J. L. HEMPSTEAD, AND T. CURRAN. 1987. Mapping patterns of c-Fos expression in the central nervous system after seizure. Science 237: 192-196. MORGAN,J. I., ANDT. CURRAN.1986. Role of ion flux in the control of c-Fos expression. Nature 322: 552-555. MORGAN, J. I., AND T. CURRAN. 1989. Calcium as a modulator of the immediate-early gene cascade in neurons. Cell Calcium 9: 303-311. MORGAN, J. I., AND T. CURRAN. 1991. Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos andjun. Annu. Rev. Neurosci. 14: 421-451. MUMENTHALER, M. 1976. The pathophysiology of pain. Pages 275-312 in (w. Birkmayer, Ed., Epileptic Seizures-BehaviourPain, Huber, Bern. NAU, H., ANDW. L&CHER. 1982. Valproic acid: Brain and plasma levels of the drug and its metabolites, anticonvulsant effects and gamma-aminobutyric acid (GABA) metabolism in the mouse. J. Phurmacol. Exp. Ther. 220: 654-659. NEDM, E., D. HEVRONI, D. NAOT, D. ISRAELI, ANDY. CITRI. 1993. Numerous car&ate plasticity-related genes revealed by differential cDNA cloning. Nature 363: 718-722. NOGUCHI,K., R. DUBNER,AND M. A. RUDA. 1992. Preproenkephalin mRNA in spinal dorsal horn neurons is induced by peripheral inflammation and is co-localized with Fos and Fos-related proteins. Neuroscience 46: 561-570. OLSEN, R. W. 1981. The GABA postsynaptic membrane receptorionophore complex. Site of action of convulsant and anticonvulsant drugs. Mol. Cell. Biochem. 39: 261-279. PATSALOS,P. N., AND P. T. LASCELLES.1981. Changes in regional brain levels of amino acid putative neurotransmitters after prolonged treatment with the anticonvulsant drugs diphenylhydantoin, phenobarbitone, sodium valproate, ethosuximide, and sulthiame in the rat. J. Neurochem. 38: 688-995. PAXINOS, G., AND C. WATSON. 1982. The rat brain in stereotaxic coordinates. Academic Press. Sydney. PERTOVAARA,A., R. BRAVO, AND T. HERDEGEN. 1993. Induction and suppression of immediate-early genes by selective alpha-aadrenoceptor agonist and antagonist in the brain following noxious peripheral stimulation. Neuroscience 54: 117-126. PRESLEY, R. W., D. MENETREZY,J. D. LEVINE, AND A I. BASLUUM. 1990. Systemic morphine suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord. J. Neurosci. 10~323-335. SATO,M., ANII F. W. FOONG.1983. A mechanism of carbamazepine analgesia as shown by bradykinii-induced trigeminal pain. Brain Res.Bull. l&407-409.

278

TGLLE

SAWAYA, M. C.B., R. W. HORTON, ANII B. S. MELDRUM. 1975. Effects of anticonvulsant drugs on the cerebral enzymes metabolizing GABA. Epilepsia IS: 649-655. 68. SAWYNOK, J. 1987. GABAergic mechanisms of analgesia: An update. Pharmacol. Biochem. Behav. 28: 463-474. 69. SCHMUTZ, M. 1985. Carbamaxepine. Pages 479-506 in H. H. Frey, and D. Janx, Eds., Handbook of Experimental Pharmacology. Antiepileptic Drugs, Vol. 74, Springer, Berlin/Heidelberg/ New York/Tokyo. 70. Scnwm, J. R., AND G. GREAT. 1989. Phenytoin and carbamaxepine: Potential- and frequency-dependent block of Na currents in mammalian myelinated nerve fibers. Epilepsia 30: 286-294. 71. S~EIT, J. E., AND C. J. WOOLF. 1985. The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord. J. Comp. Neural. 231: 66-77. 72. THEODORE, W. H. 1990. Clinical pharmacology of antiepileptic drugs. Selected topics. Neural. Clin. 8: 177-191. 73. TULLE, T. R., J. M. CASTRO-LOPES, G. EVAN, AND W. ZIEGLA~NSBERGER. 1991. c-Fos induction in the spinal cord following noxious stimulation: prevention by opiates but not by NMDA antagonist. Pages 299305 in M. R. Bond, J. E. Charlton, and C. J. Woolf, Eds., Pain Research and Clinical Management. Proc. VZth World Congress on Pain, Vol. 4. Elsevier, Amsterdam. AND W. ZIEGLG~SBERGER. 74. TI~LLE, T. R., J. M. CASTRO-LOPES, 1994a. Nociceptive stimulation induces immediate early gene expression and increases GABA immunoreactivity in spinal neurons. Pages 385-402 in T. H&felt, H.-G. Schaible, and R. F. Schmidt, Eds., Neuropeptides, Nociception and Pain. Chapman and Hall, London, Glasgow, Weinheim. 75. T~~LLE, T. R., T. HERDEGEN, J. SCHADRACK, R. BRAVO, M. ZIMMERMANN, ANDW. ZIEGLG~~SBERGER.1994b. Application of morphine prior to noxious stimulation differentially modulates expression of Fos, Jun and Krox-24 pmteins in rat spinal cord neurons. Neuroscience 54: 305-321.

67.

ET AL. 76.

G. EVAN, B. P. of Kelatorphan and morphine before and after noxious stimulation on immediateearly gene expression in rat spinal cord neurons. Pain 56: 103-112.

77.

VANDONGEN,

78. 79.

80. 81.

82. 83.

84.

85.

TULLE, T. R., J. SCHADRACK, J. M. CASTRO-LOPES. ROQUES, AND W. ZIEGLG~~SBERGER. 1994c. Effects

A. M.J.,

M. G. VANERP,

AND R. A. VOSKLNL.

1986.

Valproate reduces excitability by blockage of sodium and potassium conductance. Epilepsia 27: 177-182. WHITE, J. D., AND C. M. GALL. 1987. Differential regulation of neuropeptide and pmto-oncogene mRNA content in the hippocampus following recurrent seizures. Mol. Brain Res. 3: 21-29. WILLIAMS, S., A. PINI, G. EVAN, AND S. P. HUNT. 1989. Molecular events in the spinal cord following sensory stimulation. Page 273 in F. Cervero, G. J. Bennett, and P. M. Headley, Eds., Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord. Plenum, New York. WILSON, P. R., AND T. L. YAKSH. 1978. Baclofen is antinociceptive in the spinal intrathecal space of animals. Eur. J. Pharmacol. 51: 323-330. WISDEN, WATERS,

W., M. L. ERRINGTON, W. WILLIAMS, S. B. DIJNNE~, C. D. HITCHCOCK, G. EVAN, T. V.P. BLISS, AND S. P. HUNT.

1990. Differential expression of immediate-early hippocampus and spinal cord. Neuron 4: 603-614. ZAKRZE,WSKA,J. M. 1990. Medical management neuralgia. Br. Dent. J. 168: 399401. ZEISE,

M.

L.,

S. KASPAROW,

AND W.

genes in the of trigeminal

ZIEGLG~SBERGER.

1991.

Valproate suppresses N-methyl-D-aspartate-evoked, transient depolarizations in the rat neocortex in uitro. Brain Res. 544: 345-348. ZIEGLG~~SBERGER, W. 1986. Central control of nociception. Pages 581-656 in V. B. Mountcastle, F. E. Bloom, and S. R. Geiger, Eds., Handbook of Physiology, The Nervous System IV. Williams and Wilkins, Baltimore. ZIEGLG~~SBERGER, W., AND T. R. TULLE. 1993. The pharmacology of pain signalling. Curr. Op. Neurobiol. 3: 611-618.